This project focuses on the development of a novel vaccine strategy, HA/gp41 chimeric protein-based vaccines, for eliciting broadly neutralizing antibody responses against HIV infection. Two properties of the HIV Env underscore the difficulty in inducing broadly neutralizing antibodies, the high variability of its protein sequence even among isolates of the same clade and the steric hindrance exerted by the large, hypervariable, and heavily glycosylated gp120 subunit on conserved neutralizing epitopes. The gp41 subunit is less variable than gp120, and has been shown to contain conserved neutralizing epitopes such as those recognized by the human monoclonal antibodies 2F5, 4E10, and Z13. However, neutralizing antibodies against such conserved neutralizing epitopes in gp41 have been difficult to raise. We have in our recent studies constructed and characterized a chimeric protein HA/gp41 that it is expressed on the cell surface and exhibits enhanced reactivity to the monoclonal antibodies 2F5 and 4E10 compared to the HIV Env. Moreover, evaluation of immunogenicity in mice showed that a DNA vaccine expressing the HA/gp41 chimeric protein effectively induced gp41-specific antibody responses. Further evaluation of immunogenicity in guinea pigs showed that HA/gp41 DNA or virus-like particle (VLP) vaccines induced significant levels of gp41-specific antibody responses and these antibodies exhibit neutralizing activity against HIV Env-mediated virus infection. These observations indicate that the conserved neutralizing epitopes in the HIV gp41 can be more efficiently presented to the immune system in the setting of HA/gp41 chimeric proteins by eliminating the steric hindrance conferred by the large gp120 subunit. Furthermore, induction of HIV neutralizing antibodies suggests that the neutralizing epitopes in gp41 are presented in a proper conformation in HA/gp41 chimeric proteins. Built on these promising results, we will further develop the HA/gp41 vaccines and investigate ways to enhance their ability to induce broadly neutralizing antibodies against HIV infection. We plan to achieve our goals from two aspects. First, we will investigate whether induction of neutralizing antibodies can be augmented by an adjuvant or modifications to improve immunogenicity of the HA/gp41 vaccines (Specific Aim 1). Second, we will explore alternative approaches to modify the HA/gp41 chimeric protein and evaluate the effectiveness of these approaches for enhancing induction of neutralizing antibodies against conserved epitopes in gp41 (Specific Aim 2). Through these studies, we will gain insight to the obstacles that hinder the induction of broadly neutralizing antibodies and develop a systemic approach to tackle these difficulties for obtaining a potent HIV vaccine that induces strong broadly neutralizing antibody responses. PUBLIC HEALTH RELEVANCE: While progress has been made in the development of vaccines that elicit strong cellular immune responses, efforts to develop AIDS vaccines that induce HIV neutralizing antibody responses have met with little success, especially for the induction of cross-reactive neutralizing antibodies, which is of particular importance for an effective AIDS vaccine. The focus of this study is to further develop a novel vaccine design strategy that provide a more effctive presentation of the conserved neutralizing epitopes in gp41, with the goal to obtain a potent HIV vaccine that can induce strong and broadly HIV neutralizing antibodies